Plant histones. Relevance to the evolution of prokaryotes to eukaryotes

There are many procaryotic and eucaryotic organisms in plant kingdom. It is hoped that the study of plant histones will be useful in evolutionary studies. The histones of great variety of animal species have been studied and well characterized. Less information is available concerning plant histones. The general conclusion drawn from these investigations is that most organisms of eucaryotic plant and animal species contain the same five major histone fractions. Recently the histone-like proteins were found in some primitive eucaryotes and procaryotes. Data on histones from higher and lower eucaryotes and histone-like proteins of procaryotes are reviewed. Evolution of histones and their appearance prior to that of eucaryotic cell is postulated. The role of histones in evolution of nucleosomes is discussed.     

Putative DNA-(amino)methyltransferases in eucaryotes

By computer analysis of the known data bases, we have established that the open reading frames (ORF) coding for proteins that possess high degree of homology with procaryotic DNA-(amino)methyltransferases are present in the genomes of Leishmania major, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Arabidopsis thaliana, Drosophila melanogaster, Caenorhabditis elegans, and Homo sapiens. Conservative motifs typical for bacterial DNA-(amino)methyltransferases are detected in the amino acid sequences of these putative proteins. The ORF of all putative eucaryotic DNA-(amino)methyl-transferases found are encoded in nuclear DNA. In mitochondrial genomes including a few fully sequenced higher plant mtDNA, nucleotide sequences significantly homologous to genes of procaryotic DNA-(amino)methyltransferases are not found. Thus, ORF homologous to bacterial adenine DNA-methyltransferases are present in nuclei of protozoa, yeasts, insects, nematodes, vertebrates, higher plants, and other eucaryotes. A special search for corresponding proteins and, in particular, adenine DNA-methyltransferases in these organisms and a study of their functions are quite promising.     

Nascent secretory polypeptides synthesized on Escherichia coli ribosomes are not translocated across mammalian endoplasmic reticulum.

Cell-free protein-synthesizing systems from Escherichia coli and wheat germ were compared for their capacity to support the translocation of secretory proteins across microsomal membranes derived from mammalian endoplasmic reticulum. Three different secretory proteins, two of bacterial and one of eucaryotic origin, were tested in this respect. In all three cases a contrast between the results in the eucaryotic and procaryotic protein-synthesizing systems was revealed. Whereas the eucaryotic system, as expected, supported the translocation of nascent secretory proteins across the microsomal membranes, the procaryotic system failed to do so. This failure was not due to the absence of a translocation-promoting activity or the presence of a translocation-blocking activity in the procaryotic system. These results demonstrate a specificity in the requirement of components of the protein-synthesizing machinery for protein translocation. These components might participate in forming a functional ribosome-membrane junction during protein translocation. The nascent secretory chain alone is not sufficient for making this junction, which might involve the postulated binding of the ribosome to the signal recognition particle or another component of the membrane.     

Nucleotide sequence of the Streptococcus pneumoniae hexB mismatch repair gene: homology of HexB to MutL of Salmonella typhimurium and to PMS1 of Saccharomyces cerevisiae.

The Hex mismatch repair system of Streptococcus pneumoniae acts both during transformation (a recombination process that directly produces heteroduplex DNA) to correct donor strands and after DNA replication to remove misincorporated nucleotides. The hexB gene product is one of at least two proteins required for mismatch repair in this organism. The nucleotide sequence of a 2.7-kilobase segment from the S. pneumoniae chromosome that includes the 1.95-kilobase hexB gene was determined. The gene encodes a 73.5-kilodalton protein (649 residues). The spontaneous hex Rx chromosomal mutant allele with which a mutator phenotype has been associated is shown to result from a single base substitution (TAC to TAA) leading to a truncated HexB polypeptide (484 residues). The HexB protein is homologous to the MutL protein, which is required for methyl-directed mismatch repair in Salmonella typhimurium and Escherichia coli, and to the PMS1 gene product, which is likely to be involved in a mismatch correction system in Saccharomyces cerevisiae. The conservation of HexB-like proteins among procaryotic and eucaryotic organisms indicates that these proteins play an important common role in the repair process. This finding also suggests that the Hex, Mut, and PMS systems evolved from a common ancestor and that functionally similar mismatch repair systems could be widespread among procaryotic as well as eucaryotic organisms.     

High-efficiency transformation of Saccharomyces cerevisiae cells by bacterial minicell protoplast fusion.

After a new transformation procedure, 10% of Saccharomyces cerevisiae cells were found to contain transforming DNA sequences. We used direct transfer of plasmid molecules by fusing bacterial minicell protoplasts to yeast protoplasts. Since the procedure significantly reduces the toxic effect of procaryotic protoplasm on the eucaryotic organism, it might be generally applicable in other systems in which transformation is inefficient or impossible.     

Cancer: re-evolution of eucaryotic to procaryotic replication units--a result of genetic transposition?

We have consistently tried to present our "procaryote-hypothesis" of oncogenesis in the light of evolution. It is suggested that the replication units are the essential starting points of oncogenesis, and cancer is the result of a short and compact re-evolution from eucaryotic to procaryotic replicons. In our view, the process of re-evolution or reactivation of procaryotic replication units and/or their precursors starts with the activity of a cancerogen and proceeds by genetic rearrangements, possibly by genetic transposition. Consequently, some remarks had to be made on: (a) the evolution of self-replicative species; (b) the evolvement of procaryotes to eucaryotes; and (c) the various mobile repeated genetic elements. Moreover, we have taken into consideration the important fact that most of the mammalian genome is reversibly repressed and "that there is in these modern genes still a memory of ancient sequences" ( Eigen et al., 1981). We can only speculate on the extent of the evolutionary conservation, particularly of certain ancient nucleotide sequences related to autonomous, indefinite self-replication. Nevertheless, no real argument can be made against the simple possibility that a re-evolution of eucaryotic to procaryotic replicons lead to cancer.     

Commercial exploitation of transgenics

A series of examples of the application of recombinant nucelic acid technology to the production of transgenic organisms is presented. The review considers the distinct advantages of producing certain recombinant biomolecules in appropriate eucaryotic systems rather than in the traditional procaryotic ones. Gene therapy in humans and the production of transgenic animals and plants are discussed. Concerns about the inadvertant construction of new pathogens and ethical considerations about interfereing with poorly understood ecosystems are addressed.     

Procaryotic genomic DNA inhibits mammalian cell transformation.

Ltk- mouse cells were transformed to thymidine kinase prototrophy in the presence of carrier DNAs isolated from different organisms. Procaryotic genomic and phage DNA was consistently less effective as a carrier than was eucaryotic DNA. Mixing experiments indicate that DNA of procaryotic origin inhibits mammalian cell transformation.     

Size comparisons among integral membrane transport protein homologues in bacteria, Archaea, and Eucarya

Integral membrane proteins from over 20 ubiquitous families of channels, secondary carriers, and primary active transporters were analyzed for average size differences between homologues from the three domains of life: Bacteria, Archaea, and Eucarya. The results showed that while eucaryotic homologues are consistently larger than their bacterial counterparts, archaeal homologues are significantly smaller. These size differences proved to be due primarily to variations in the sizes of hydrophilic domains localized to the N termini, the C termini, or specific loops between transmembrane alpha-helical spanners, depending on the family. Within the Eucarya domain, plant homologues proved to be substantially smaller than their animal and fungal counterparts. By contrast, extracytoplasmic receptors of ABC-type uptake systems in Archaea proved to be larger on average than those of their bacterial homologues, while cytoplasmic enzymes from different organisms exhibited little or no significant size differences. These observations presumably reflect evolutionary pressure and molecular mechanisms that must have been operative since these groups of organisms diverged from each other.     

查尔酮合酶基因对转基因植物花色和育性的影响

查尔酮合酶 ( chalcone synthase,CHS)是花色素合成途径中的一个关键酶 ,它在植物中表达量的改变可能影响花的颜色。从矮牵牛 ( Petunia hybrida)特定发育阶段的花瓣的 c DNA中 ,克隆到查尔酮合酶基因 ,并正向插入到原核表达载体和含有花椰菜花叶病毒 Ca MV 35 S启动子的真核表达载体中 ,在原核中得到高效表达 ,并通过土壤农杆菌介导的方法转化矮牵牛。转基因植物的花色不但发生了明显的变异 ,其育性也受到了影响 ,不能产生正常花粉粒 ,成为雄性不育植株。 Northern杂交表明 ,转基因植物花瓣中 ,内源及外源查尔酮合酶基因转录均受到抑制     

Biochemical studies on the mutagen, 6-N-hydroxylaminopurine. Synthesis of the deoxynucleoside triphosphate and its incorporation into DNA in vitro

The nucleotide analogue, 6-N-hydroxylaminopurine deoxynucleoside triphosphate (dHAPTP) has been synthesized from 6-chloropurine by a procedure involving both enzymatic and chemical reagents. In a series of experiments involving several different DNA polymerases including 3 procaryotic and 2 eucaryotic enzymes, it was shown that dHAPTP is ambiguous in its base-pairing characteristics, since it can replace both dATP and dGTP in DNA synthesis. It was also shown that different enzymes have different capacities to distinguish dHAPTP from the canonical deoxynucleoside triphosphates. These results are consistent with (but do not prove) the hypothesis that the mechanism of 6-N-hydroxylaminopurine mutagenesis seen in both eucaryotic and procaryotic organisms is due to its conversion, in vivo, to a deoxynucleoside triphosphate which is incorporated ambiguously for dATP and dGTP during DNA replication.     

Structure and function of initiator methionine tRNA from the mitochondria of Neurospora crassa.

Initiator methionine tRNA from the mitochondria of Neurospora crassa has been purified and sequenced. This mitochondrial tRNA can be aminoacylated and formylated by E. coli enzymes, and is capable of initiating protein synthesis in E. coli extracts. The nucleotide composition of the mitochondrial initiator tRNA (the first mitochondrial tRNA subjected to sequence analysis) is very rich in A + U, like that reported for total mitochondrial tRNA. In two of the unique features which differentiate procaryotic from eucaryotic cytoplasmic initiator tRNAs, the mitochondrial tRNA appears to resemble the eucaryotic initiator tRNAs. Thus unlike procaryotic initiator tRNAs in which the 5′ terminal nucleotide cannot form a Watson-Crick base pair to the fifth nucleotide from the 3′ end, the mitochondrial tRNA can form such a base pair; and like the eucaryotic cytoplasmic initiator tRNAs, the mitochondrial initiator tRNA lacks the sequence -TΨCG(or A) in loop IV. The corresponding sequence in the mitochondrial tRNA, however, is -UGCA- and not -AU(or Ψ)CG-as found in all eucaryotic cytoplasmic initiator tRNAs. In spite of some similarity of the mitochondrial initiator tRNA to both eucaryotic and procaryotic initiator tRNAs, the mitochondrial initiator tRNA is basically different from both these tRNAs. Between these two classes of initiator tRNAs, however, it is more homologous in sequence to procaryotic (56–60%) than to eucaryotic cytoplasmic initiator tRNAs (45–51%).     

Sex-chromosome evolution: recent progress and the influence of male and female heterogamety

It is now clear that sex chromosomes differ from autosomes in many aspects of genome biology, such as organization, gene content and gene expression. Moreover, sex linkage has numerous evolutionary genetic implications. Here, I provide a coherent overview of sex-chromosome evolution and function based on recent data. Heteromorphic sex chromosomes are almost as widespread across the animal and plant kingdoms as sexual reproduction itself and an accumulating body of genetic data reveals interesting similarities, as well as dissimilarities, between organisms with XY or ZW sex-determination systems. Therefore, I discuss how patterns and processes associated with sex linkage in male- and female-heterogametic systems offer a useful contrast in the study of sex-chromosome evolution.     

Transgenic Plants: New Biological System or New Properties of Plant-<Emphasis Type="Italic">Agrobacterium</Emphasis> Symbiosis?

Transgenic plants have become one of the most important subjects of inquiry in biotechnology. Disputes about possible consequences of the use of genetically modified organisms still continue. Biological status of genetically modified plants is unsettled. Common criteria for assessment of the consequences of genetic transformation are lacking. Causes for and mechanisms of unforeseen consequences of transformation remain obscure. Methods of genetic engineering of plants are based on plant-Agrobacterium interaction. Transgenic plants are regarded as super-species systems produced as a result of artificial plant-Agrobacterium symbioses. Mechanisms of interaction between the members of symbiosis are very intricate and labile. Incorporation of foreign genes into T-DNA modifies characteristics of a bacterial symbiont. As a result, interaction between the organisms changes. The systems of inherent plant immunity are activated and plant metabolism is accordingly reorganized. These changes are the main reason for unintended effects of transgenosis. The intensity of response to transformation depends on the characteristics of the inserted target gene.     

Gene transfer and gene mapping in mammalian cells in culture

The ability to transfer mammalian genes parasexually has opened new possibilities for gene mapping and fine structure mapping and offers great potential for contributing to several aspects of mammalian biology, including gene expression and genetic engineering. The DNA transferred has ranged from whole genomes to single genes and smaller segments of DNA. The transfer of whole genomes by cell fusion forms cell hybrids, which has promoted the extensive mapping of human and mouse genes. Transfer, by cell fusion, of rearranged chromosomes has contributed significantly to determining close linkage and the assignment of genes to specific chromosomal regions. Transfer of single chromosomes has been achieved utilizing microcells fused to recipient cells. Metaphase chromosomes have been isolated and used to transfer single-to-multigenic DNA segments. DNA-mediated gene transfer, simulating bacterial transformation, has achieved transfer of single-copy genes. By utilizing DNA cleaved with restriction endonucleases, gene transfer is being empolyed as a bioassay for the purification of genes. Gene mapping and the fate of transferred genes can be examined now at the molecular level using sequence-specific probles. Recently, single genes have been cloned into eucaryotic and procaryotic vectors for transfer into mammalian cells. Moreover, recombinant libraries in which entire mammalian genomes are represented collectively are a rich new source of transferable genes. Methodology for transferring mammalian genetic information and applications for mapping mammalian genes is presented and prospects for the future discussed.     

The use of detergent-based aqueous two-phase systems for the isolation of extracellular proteins: purification of a lipase from Pseudomonas cepacia.

The partitioning of a variety of extracellular lipases, both pro- and eucaryotic, in detergent-based aqueous two-phase systems was examined. The results revealed that all procaryotic lipases showed a clear preference for the detergent-rich coacervate phase. In contrast, all eucaryotic lipases were significantly excluded from this phase, most probably caused by their glycosylation. The potential of such detergent-based systems for the isolation of extracellular lipases directly from cell-free culture broth was analyzed using the bacterium Pseudomonas cepacia (DSM 50181). This strain was identified after a limited screening for lipase activity. About 76% of the lipase could be extracted into the coacervate phase in just one purification step, leading to a four-fold concentration of lipase and a purification factor of 24.     

Cloning and expression in Escherichia coli of isopenicillin N synthetase genes from Streptomyces lipmanii and Aspergillus nidulans.

beta-Lactam antibiotics such as penicillins and cephalosporins are synthesized by a wide variety of microbes, including procaryotes and eucaryotes. Isopenicillin N synthetase catalyzes a key reaction in the biosynthetic pathway of penicillins and cephalosporins. The genes encoding this protein have previously been cloned from the filamentous fungi Cephalosporium acremonium and Penicillium chrysogenum and characterized. We have extended our analysis to the isopenicillin N synthetase genes from the fungus Aspergillus nidulans and the gram-positive procaryote Streptomyces lipmanii. The isopenicillin N synthetase genes from these organisms have been cloned and sequenced, and the proteins encoded by the open reading frames were expressed in Escherichia coli. Active isopenicillin N synthetase enzyme was recovered from extracts of E. coli cells prepared from cells containing each of the genes in expression vectors. The four isopenicillin N synthetase genes studied are closely related. Pairwise comparison of the DNA sequences showed between 62.5 and 75.7% identity; comparison of the predicted amino acid sequences showed between 53.9 and 80.6% identity. The close homology of the procaryotic and eucaryotic isopenicillin N synthetase genes suggests horizontal transfer of the genes during evolution.     

Double-stranded RNA

High molecular weight, fully double-stranded RNA (dsRNA) has been recognized as the genetic material of many plant, animal, fungal, and bacterial viruses (Diplornaviruses): virusspecific dsRNA is also found in cells infected with single-stranded RNA viruses. DsRNA has identified in a variety of apparently normal eucaryotic cells and is associated with the "killer" character of certain strains of Saccaromyces cerevisiae.